Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

Protein translocase of mitochondrial inner membrane in Trypanosoma brucei

Translocases of mitochondrial inner membrane (TIMs) are multiprotein complexes. The only Tim component so far characterized in kinetoplastid parasites such as Trypanosoma brucei is Tim17 (TbTim17), which is essential for cell survival and mitochondrial protein import. Here, we report that TbTim17 is present in a protein complex of about 1,100 kDa, which is much larger than the TIM complexes found in fungi and mammals. Depletion of TbTim17 in T. brucei impairs the mitochondrial import of cytochrome oxidase subunit IV, an N-terminal signal-containing protein. Pretreatment of isolated mitoplasts with the anti-TbTim17 antibody inhibited import of cytochrome oxidase subunit IV, indicating a direct involvement of the TbTim17 in the import process. Purification of the TbTim17-containing protein complex from the mitochondrial membrane of T. brucei by tandem affinity chromatography revealed that TbTim17 associates with seven unique as well as a few known T. brucei mitochondrial proteins. Depletion of three of these novel proteins, i.e. TbTim47, TbTim54, and TbTim62, significantly decreased mitochondrial protein import in vitro. In vivo targeting of a newly synthesized mitochondrial matrix protein, MRP2, was also inhibited due to depletion of TbTim17, TbTim54, and TbTim62. Co-precipitation analysis confirmed the interaction of TbTim54 and TbTim62 with TbTim17 in vivo. Overall, our data reveal that TbTim17, the single homolog of Tim17/22/23 family proteins, is present in a unique TIM complex consisting of novel proteins in T. brucei and is critical for mitochondrial protein import.     

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.     

Calcium depletion and calmodulin inhibition affect the import of nuclear-encoded proteins into plant mitochondria

Many metabolic processes essential for plant viability take place in mitochondria. Therefore, mitochondrial function has to be carefully balanced in accordance with the developmental stage and metabolic requirements of the cell. One way to adapt organellar function is the alteration of protein composition. Since most mitochondrial proteins are nuclear encoded, fine-tuning of mitochondrial protein content could be achieved by the regulation of protein translocation. Here we present evidence that the import of nuclear-encoded mitochondrial proteins into plant mitochondria is influenced by calcium and calmodulin. In pea mitochondria, the calmodulin inhibitor ophiobolin A as well as the calcium ionophores A23187 and ionomycin inhibit translocation of nuclear-encoded proteins in a concentration-dependent manner, an effect that can be countered by the addition of external calmodulin or calcium, respectively. Inhibition was observed exclusively for proteins translocating into or across the inner membrane but not for proteins residing in the outer membrane or the intermembrane space. Ophiobolin A and the calcium ionophores further inhibit translocation into mitochondria with disrupted outer membranes, but their effect is not mediated via a change in the membrane potential across the inner mitochondrial membrane. Together, our results suggest that calcium/calmodulin influences the import of a subset of mitochondrial proteins at the inner membrane. Interestingly, we could not observe any influence of ophiobolin A or the calcium ionophores on protein translocation into mitochondria of yeast, indicating that the effect of calcium/calmodulin on mitochondrial protein import might be a plant-specific trait.     

Mitochondrial membrane complex that contains proteins necessary for tRNA import in Trypanosoma brucei

The mitochondrial genome of Trypanosoma brucei does not contain genes encoding tRNAs; instead this protozoan parasite must import nuclear-encoded tRNAs from the cytosol for mitochondrial translation. Previously, it has been shown that mitochondrial tRNA import requires ATP hydrolysis and a proteinaceous mitochondrial membrane component. However, little is known about the mitochondrial membrane proteins involved in tRNA binding and translocation into the mitochondrion. Here we report the purification of a mitochondrial membrane complex using tRNA affinity purification and have identified several protein components of the putative tRNA translocon by mass spectrometry. Using an in vivo tRNA import assay in combination with RNA interference, we have verified that two of these proteins, Tb11.01.4590 and Tb09.v1.0420, are involved in mitochondrial tRNA import. Using Protein C Epitope -Tobacco Etch Virus-Protein A Epitope (PTP)-tagged Tb11.01.4590, additional associated proteins were identified including Tim17 and other mitochondrial proteins necessary for mitochondrial protein import. Results presented here identify and validate two novel protein components of the putative tRNA translocon and provide additional evidence that mitochondrial tRNA and protein import have shared components in trypanosomes.     

Thyroid hormone regulation of nuclear-encoded mitochondrial inner membrane polypeptides of the liver

The effects of thyroid hormone on nuclear-encoded mitochondrial inner membrane proteins were investigated by in vitro translation of the endogenous mRNA present in a postmitochondrial fraction from the livers of rats treated in vivo with hormone. The levels of the mRNAs were estimated by quantitative immunoabsorption of the translation mixture. Total protein synthesis was increased 2.6-fold after 4 days of in vivo hormone treatment, but only 10-15% of the polypeptides were dramatically altered (greater than 5-fold). Among the most highly elevated were cytochrome c1 (greater than 10-fold increase) and the Rieske iron-sulfur protein of the cytochrome bc1 complex. Other inner membrane proteins (core protein 1, beta subunit of F1 ATPase, subunit IV of cytochrome oxidase, 3-hydroxybutyrate dehydrogenase) and non-mitochondrial proteins (rat serum albumin, beta 2-microglobulin) were not altered significantly by hormone treatment. Cytochrome c1 and the Rieske protein increased after 12 h of hormone treatment, a relatively early response in mammalian mitochondrial biogenesis. The possible significance of this response for the regulation of mitochondrial synthesis and assembly is discussed.     

Events upstream of mitochondrial protein import limit the oxidative capacity of fibroblasts in multiple mitochondrial disease

To investigate whether protein import is defective in mitochondrial disease, we compared the rate of import and the expression of protein import machinery components in skin fibroblasts from control subjects and a patient with multiple mitochondrial disease (MMD). The patient exhibited a 35% decrease in cytochrome c oxidase activity and a 59% decrease in cellular oxygen consumption compared to control. Western blot analyses revealed that patient levels of MDH, mtHSP70, HSP60, and Tom20 protein were 57%, 20%, 75% and 100% of control cells, respectively. MDH and Tom20 mRNA levels were not different from control levels, whereas mtHSP70 mRNA were 50% greater than control. Radiolabeled MDH was imported into mitochondria with equal efficiency between patient (44% of total synthesized) and control (43%) cells, although the total MDH synthesized in patient cells was reduced by about 40%. The unaffected levels of mRNA and post-translational import into mitochondria, combined with reduced protein levels of MDH, mtHSP70, and HSP60 suggest a translational defect in this patient with MMD. This was verified by the 50% reduction in overall cellular protein synthesis in the patient compared to control. Further, the similar import rates between patient and control cells suggest an important role for Tom20, but a lesser role for mtHSP70 in regulating protein import into mitochondria.     

Insulin-resistant muscle is exercise resistant: evidence for reduced response of nuclear-encoded mitochondrial genes to exercise

Mitochondrial dysfunction, associated with insulin resistance, is characterized by low expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and nuclear-encoded mitochondrial genes. This deficit could be due to decreased physical activity or a decreased response of gene expression to exercise. The objective of this study was to investigate whether a bout of exercise induces the same increase in nuclear-encoded mitochondrial gene expression in insulin-sensitive and insulin-resistant subjects matched for exercise capacity. Seven lean and nine obese subjects took part. Insulin sensitivity was assessed by an 80 mU.m(-2).min(-1) euglycemic clamp. Subjects were matched for aerobic capacity and underwent a single bout of exercise at 70 and 90% of maximum heart rate with muscle biopsies at 30 and 300 min postexercise. Quantitative RT-PCR and immunoblot analyses were used to determine the effect of exercise on gene expression and protein abundance and phosphorylation. In the postexercise period, lean subjects immediately increased PGC-1alpha mRNA level (reaching an eightfold increase by 300 min postexercise) and protein abundance and AMP-dependent protein kinase phosphorylation. Activation of PGC-1alpha was followed by increase of nuclear respiratory factor-1 and cytochrome c oxidase (subunit VIc). However, in insulin-resistant subjects, there was a delayed and reduced response in PGC-1alpha mRNA and protein, and phosphorylation of AMP-dependent protein kinase was transient. None of the genes downstream of PGC-1alpha was increased after exercise in insulin resistance. Insulin-resistant subjects have a reduced response of nuclear-encoded mitochondrial genes to exercise, and this could contribute to the origin and maintenance of mitochondrial dysfunction.     

Import Pathway of Nuclear-Encoded Cytochrome c Oxidase Subunit 2 Using Yeast as a Model

Abstract: Subunit 2 of cytochrome c oxidase (Cox2) is a mitochondrial-encoded protein in most organisms. In soybean Glycine max a second Cox2 gene was identified in the nucleus which is functional, whereas the mitochondrial-encoded cox2 gene is silent. For import and sorting of the nuclear-encoded soybean Cox2 protein ( Gm Cox2p) into mitochondria, the protein has acquired an N-terminal extension of 136 amino acid residues that is cleaved off in three steps during import. To study the function and processing of the Gm Cox2p leader peptide, we used yeast as a model system. Using different leader peptide-GFP constructs, we were able to show that the i₁ intermediate is generated in the mitochondrial matrix and the mature protein is generated in the inner membrane space. Mitochondrial processing peptidase (MPP) is involved in processing the first part of the leader peptide, processing of the last part is catalysed by the inner membrane peptidase (IMP). Oxa1p is necessary for insertion of the protein into the inner mitochondrial membrane. Gm Cox2p therefore utilises many of the same components as its mitochondrial-encoded predecessor, for sorting and maturation, following its import into the mitochondria.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigating the role of the physiological isoform switch of cytochrome c oxidase subunits in reversible mitochondrial disease

Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues.We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Mitochondrial mRNA localization is governed by translation kinetics and spatial transport

For many nuclear-encoded mitochondrial genes, mRNA localizes to the mitochondrial surface co-translationally, aided by the association of a mitochondrial targeting sequence (MTS) on the nascent peptide with the mitochondrial import complex. For a subset of these co-translationally localized mRNAs, their localization is dependent on the metabolic state of the cell, while others are constitutively localized. To explore the differences between these two mRNA types we developed a stochastic, quantitative model for MTS-mediated mRNA localization to mitochondria in yeast cells. This model includes translation, applying gene-specific kinetics derived from experimental data; and diffusion in the cytosol. Even though both mRNA types are co-translationally localized we found that the steady state number, or density, of ribosomes along an mRNA was insufficient to differentiate the two mRNA types. Instead, conditionally-localized mRNAs have faster translation kinetics which modulate localization in combination with changes to diffusive search kinetics across metabolic states. Our model also suggests that the MTS requires a maturation time to become competent to bind mitochondria. Our work indicates that yeast cells can regulate mRNA localization to mitochondria by controlling mitochondrial volume fraction (influencing diffusive search times) and gene translation kinetics (adjusting mRNA binding competence) without the need for mRNA-specific binding proteins. These results shed light on both global and gene-specific mechanisms that enable cells to alter mRNA localization in response to changing metabolic conditions.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.